The present invention relates to a method for detecting fetal Down syndrome (Trisomy 21) during prenatal screening. This method also relates to other more rare but detectable chromosomal trisomies such as Trisomy 13 and Trisomy 18. More particularly the present invention relates to a method for improving detection efficiency in screening for Down syndrome by measuring the amount of the free beta subunit of human chorionic gonadotropin (hCG) in the blood of pregnant women.
Down syndrome, also referred to as Trisomy 21, is the most common congenital cause of severe mental retardation. Generally, fetal Down syndrome can be determined by a diagnostic procedure including amniocentesis and karyotyping. However, this diagnostic procedure is invasive and involves risk to the woman and the fetus. Amniocentesis and karyotyping are not routinely performed during all pregnancies. Instead, one or more screening methods may be utilized to determine when the risk to the pregnancy warrants the risk of undergoing an invasive diagnostic procedure.
The incidence of Down syndrome increases significantly with increasing maternal age. Historically, the prenatal search for Down syndrome has focused on pregnant women at and over the age of 35, at which ages the risks of Down syndrome approach or exceed the risks of diagnostic procedures utilized to detect fetal Down syndrome. Therefore the standard method of prenatal screening has involved selecting women for diagnostic amniocentesis on the basis of maternal age. Age, however, is an inadequate screening criterion in that only about 20% of all Down syndrome pregnancies can be detected by carrying out amniocentesis and karyotyping on the 5% of pregnant women most at risk, that is, those aged 35 years or greater. And, because in actual clinical practice only about half of the women aged 35 years or greater undergo amniocentesis and karyotyping, fewer than 10% of Down syndrome pregnancies are prenatally detected.
In 1984 an association between lowered maternal blood alpha-fetoprotein (AFP) levels and fetal Down syndrome was discovered. For example, see "An association between low maternal serum alpha-fetoprotein and fetal chromosomal abnormalities"; Merkatz, Macri, et al.; Am. J. Obstet. Gynecol. 148:886, 1984; the disclosure of which is hereby incorporated by reference. In this publication it was noted that other chromosomal trisomies, in particular Trisomy 13 and Trisomy 18, were also associated with lowered maternal blood AFP levels. The incidence of these additional chromosomal trisomies (1 in 5000 pregnancies and 1 in 6600 pregnancies, respectively) is significantly lower than the general a priori risk associated with Trisomy 21 (Down syndrome). However, because of the association of these other chromsomal trisomies with lowered MSAFP levels, such abnormalities will also be detected within a screening protocol utilizing maternal blood AFP and the free beta subunit of hCG and possibly additional markers described herein. It is obvious to those skilled in the art that in using the protocol described herein for Trisomy 21, the detection of Trisomy 13 and 18 may also be accomplished. The association between lowered maternal blood AFP levels and fetal Down syndrome presented the opportunity to use a non-invasive blood screening test in the detection of Down syndrome cases in young, apparently unaffected families where approximately 80% of Down syndrome cases occur. It is estimated that the use of a screening test based on low maternal blood AFP (as a screening marker) would lead to the prenatal detection of approximately 20% of all cases of fetal Down syndrome.
Another method for screening involves measuring the level of unconjugated estriol (UE) in maternal blood. For example, see "Maternal blood screening for Down syndrome in early pregnancy"; Wald, et al. British Journal of Obstetrics and Gynocology (BMJ) Volume 95, April 1988, the disclosure of which is hereby incorporated by reference. The measurement of UE however, provides a poor basis for screening.
More recently an association between elevated maternal blood hCG levels, elevated maternal blood level of the alpha subunit of hCG (hCG is composed of two subunits, hereinafter referred to as alpha-hCG and beta-hCG respectively), and fetal Down syndrome was discovered. For example, see "Abnormal Maternal Serum Chorionic Gonadotropin Levels in Pregnancies with Fetal Chromosome Abnormalities"; Bogart, Pandian and Jones; Prenatal Diagnosis, Vol. 7, 623-630 (1987) the disclosure of which is hereby incorporated by reference. In the Bogart article it is estimated that the use of elevated maternal blood hCG levels and elevated maternal blood levels of the alpha subunit of hCG, would detect approximately 68% of the chromosomally abnormal fetuses. However, these results were obtained from a study on pregnancies at 18-25 weeks of gestation and the affected cases appear to be of women previously identified as being at risk for Down syndrome.
Generally, as suggested above, screening by evaluation of maternal blood hCG has involved only the measurement of hCG in general and additionally the measurement of alpha-hCG. Although these screening methods do detect fetal Down syndrome, there is a need and a desire for a method which detects a greater percentage of fetal Down syndrome cases.
I have discovered a previously unknown association between elevated levels of maternal blood free beta-hCG and fetal Down syndrome. I have also discovered a previously unknown association between the maternal blood level of free beta-hCG and the maternal blood level of AFP and fetal Down syndrome, I have further discovered a previously unknown association between the ratio of the maternal blood level of free beta-hCG to the maternal blood level of the intact hCG molecule and fetal Down syndrome. I have still further discovered that using a multivariate discriminant analysis technique improves the detection efficiency of a screening method using the maternal blood level of free beta-hCG, or the maternal blood level of free beta-hCG and the maternal blood level of AFP, or the log of either, or the log of both, especially when gestational age is also incorporated as a variable in the discriminant analysis technique, for a chosen risk cut-off level. Gestational age refers to the age of the pregnant woman's fetus. Detection efficiency refers to the percentage of cases of fetal Down syndrome which are correctly detected for a chosen risk cut off level. The risk cut off level will be more fully explained in a following section. Discriminant analysis is a generally known approach to multivariate analysis involving the separation of a population into two or more groups by a univariate risk assessment. Discriminant analysis is also sometimes described as a way of constructing a linear combination of independent variables, thus reducing the problem of measuring group differences to a univariate problem. Discriminant analysis can also be performed when there is only one variable involved in a problem. A general discussion of discriminant analysis can be found in Marketing Research; Churchill, G. A.; Dryden, 1976; Chapter 15, pages 530-543, the disclosure of which is hereby incorporated by reference. I have discovered that subjecting the maternal blood levels of free beta-hCG, the maternal blood levels of intact hCG, the ratio of the maternal blood level of free beta-hCG to the maternal blood level of the intact hCG molecule, the maternal blood level of AFP, the maternal blood level of UE, and gestational age to multi-variate discriminant analysis detects a greater percentage, with a lower false positive rate, of fetal Down syndrome cases than any other known screening method for the prenatal detection of Down syndrome. I have further discovered that a still greater number of the cases of fetal Down syndrome may be detected by using only the measurements of the maternal blood levels of free-beta hCG and the maternal blood levels of AFP and subjecting the log of each measurement and gestational age to a multivariate discriminant analysis. These and other discoveries will be more fully explained in the Summary of the Invention section and the Detailed Description of the Invention conventional immunological methods which can include immunoassay techniques such as those referred to in the papers above, and other techniques known in the art. The level of free beta-hCG is then compared to a set of reference data to determine the patient's risk of carrying a fetus with Down syndrome. To improve detection efficiency, the level of free beta-hCG and the gestational age can be compared to a set of reference data. To further improve detection efficiency, a patient's maternal blood levels of free beta-hCG and AFP (referred to as "markers") are measured by conventional immunological methods, including assay techniques known to the art such as those referred to in the papers above. The levels of each marker are then compared to a set of reference data to determine the patient's risk of carrying a fetus with Down syndrome. A multivariate discriminant analysis technique is used to compare the levels of the markers to a set of reference data. More particularly, a patient specific risk is then calculated using Bayes rule, the patient's a priori risk, and the relative frequencies for unaffected and affected pregnancies which are determined by incorporating the log of the patient's quantitative levels of each marker into the probability density functions for the reference data developed using multivariate discriminant analysis. If the patient's risk of carrying a fetus with Down syndrome is greater than a given risk cut-off level, the patient should be counseled about further diagnostic tests to confirm the presence of Down syndrome. Incorporating gestational age as a marker along with the level of free beta-hCG and the maternal blood level of AFP will further improve detection efficiency. Since the maternal blood level of free beta-hCG and the maternal blood level of AFP for a number of samples tend to be distributed according to a log-gaussian distribution curve, the greatest detection efficiency can be achieved by incorporating the log of the patient's quantitative levels of each marker and gestational age into the probability density functions for the reference data developed using multivariate discriminant analysis.
An advantage of the method and process of the present invention is that it correctly predicts a higher percentage of fetal Down syndrome cases, with a lesser false positive rate than other known methods and processes.
Other advantages of the present invention will become clear from the following more detailed description and the following examples.